Normal cell development requires the interaction of RNA polymerase with many factors that regulate transcriptional activity. We study a simple phage system, regulation of bacteriophage T4 middle promoters, as a model for how factors can change the specificity of RNA polymerase. Middle promoters are recognized by host RNA polymerase in vitro, and they contain sequences in the -10 region that are recognized by the primary specificity subunit of E. coli RNA polymerase, the sigma 70 subunit. However, middle transcription does not begin until about 1 minute post-infection, because phage modifications to the polymerase immediately after infection inhibit this ability of RNA polymerase alone to transcribe from these promoters. In order to transcribe from middle promoters, the phage-modified polymerase requires both a phage-encoded activator, MotA, and a phage-encoded coactivator, AsiA. Previously, we have shown that both MotA and AsiA bind independently to sigma 70. Our DNase I footprints of the T4 middle promoter PuvsX have revealed striking differences upstream of position -20 in the binding of unmodified polymerase alone versus the polymerase/AsiA/MotA complex. Our recent results suggest that the binding of the MotA/AsiA/polymerase complex requires sigma's C-terminal 43 amino acids and the N-terminal region of MotA. We have demonstrated that MotA also binds to a promoter sequence centered at -30 (a MotA box), but that this binding is relatively weak with an apparent dissociation constant of about 100 nM. Competition assays indicate that there is only about a 10-fold difference in MotA affinity for a wild type consensus sequence (TTTGCTTCA) versus one lacking a recognizable MotA box. This result suggests flexible sequence requirements for MotA binding. Our primer extension analyses of MotA-dependent transcripts from a T4 infection have revealed new MotA box sequences in addition to the previously derived MotA consensus sequence, confirming these results in vivo. We suggest that in middle promoters the architecture of the -35 region is redesigned by the binding of AsiA and MotA to the C-terminal domain of sigma, and that the weak binding by MotA to the DNA is stabilized by its interaction with sigma 70. Thus, this system provides a model for investigating how protein-protein contacts among a polymerase, an activator, and a coactivator can result in transcriptional activation.